Few Layer Ti3C2 MXene-Based Label-Free Aptasensor for Ultrasensitive Determination of Chloramphenicol in Milk

Quantitative detection of veterinary drug residues in animal-derived food is of great significance. In this work, a simple and label-free electrochemical aptasensor for the highly sensitive detection of chloramphenicol (CAP) in milk was successfully developed based on a new biosensing method, where the single- or few-layer Ti3C2 MXene nanosheets functionalized via the specific aptamer by self-assembly were used as electrode modifiers for a glassy carbon electrode (aptamer/Ti3C2 MXene/GCE). Differential pulse voltammetry (DPV), electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), scanning electron microscopy (SEM), atomic force microscope (AFM), and so on were utilized for electrochemical and morphological characterization. Under the optimized conditions, the constructed aptasensor exhibited excellent performance with a wider linearity to CAP in the range from 10 fM to 1 μM and a low detection limit of 1 fM. Aptamer/Ti3C2 MXene/GCE demonstrated remarkable selectivity over other potentially interfering antibiotics, as well as exceptional reproducibility and stability. In addition, the aptasensor was successfully applied to determine CAP in milk with acceptable recovery values of 96.13% to 108.15% and relative standard deviations below 9%. Therefore, the proposed electrochemical aptasensor is an excellent alternative for determining CAP in food samples.


Introduction
Animal-derived foods such as milk and eggs are an important part of daily diets. With the widespread use of veterinary drugs for the treatment and prevention of diseases in animals and the promotion of animal growth, the possible presence of veterinary drug residues in animal-derived foods is one of the key issues for food safety. Chloramphenicol (CAP), produced by Streptomyces Venezuelan [1], is an effective broad-spectrum antibiotic normally used in animals for treating and preventing infectious diseases [2]. However, excessive use of CAP can result in inevitable residues in animal-derived food, which subsequently bring about serious toxic side effects on human health through the food chain, such as aplastic anemia, gray baby syndrome, leukemia, nausea, diarrhea, and allergic reactions [3]. To date, various analytical methods, including high-performance liquid chromatography [4], liquid chromatography-mass spectrometry [5], and gas chromatographymass spectrometry [6], have been developed for quantitively determining CAP in food samples. Although these methods are accurate and reliable, they have some inevitable limitations, such as their time-consuming nature, high cost, tedious operation procedures, professional operation skills, and excessive dependence on expensive and sophisticated instruments, all of which impede their application in the real-time monitoring of CAP. In addition, normally used microbial assays and enzyme-linked immuno-sorbent assays also have disadvantages [7,8], including low sensitivity, false positives, and cross-reactions. Otherwise, because they are simple, fast, and highly sensitive, various biosensing strategies In this work, a simple Ti 3 C 2 MXene-based electrochemical aptasensor with high sensitivity, selectivity, and stability for label-free detection of CAP in milk has been put forward, as shown in Figure 1. The stacking and aggregation of Ti 3 C 2 MXene by van der Waals forces showed weaker electrochemical performance, and on the contrary, single-or fewer-layer Ti 3 C 2 MXene nanosheets could provide more electron transport channels as well as more surface binding sites and a specific interface. When the aptamer specifically binds to CAP, electronic channels reduce, which results in a decrease in the response current related to the concentration of CAP. In addition, selectivity, reproducibility, and stability studies were carried out and obtained results that indicated that aptamer/Ti 3 C 2 MXene/glassy carbon electrode (GCE) had shown an anti-interference property with longterm stability. The feasibility of the proposed aptasensor was verified by the analysis of real milk samples, which suggested that it was a new sensing platform for the detection of CAP in food samples. studies indicating that ssDNA may adsorb on the surface of Ti3C2 MXene nanosheets through π-π stacking between the aromatic nucleobases of DNA and graphite structure of MXene [32].
In this work, a simple Ti3C2 MXene-based electrochemical aptasensor with high sensitivity, selectivity, and stability for label-free detection of CAP in milk has been put forward, as shown in Figure 1. The stacking and aggregation of Ti3C2 MXene by van der Waals forces showed weaker electrochemical performance, and on the contrary, single-or fewer-layer Ti3C2 MXene nanosheets could provide more electron transport channels as well as more surface binding sites and a specific interface. When the aptamer specifically binds to CAP, electronic channels reduce, which results in a decrease in the response current related to the concentration of CAP. In addition, selectivity, reproducibility, and stability studies were carried out and obtained results that indicated that aptamer/Ti3C2 MXene/glassy carbon electrode (GCE) had shown an anti-interference property with longterm stability. The feasibility of the proposed aptasensor was verified by the analysis of real milk samples, which suggested that it was a new sensing platform for the detection of CAP in food samples.

Characterization of Ti3C2 MXene
The X-ray diffraction (XRD) features of Ti3AlC2 and Ti3C2 MXene are shown in Figure 2A. The (002) peak of Ti3C2 MXene shifted to a lower angle as compared to the commercial Ti3AlC2 phase, suggesting a successful removal of Al layers after HF exfoliation [33]. The appearance of the Ti3C2 MXene peak at approximately 25° was attributed to the (104) diffraction of TiO2, which demonstrated a certain amount of oxides. The morphology and structure of Ti3C2 MXene film were characterized by atomic force microscope (AFM) ( Figure S1A,B) and scanning electron microscopy (SEM) ( Figure 2D). Figure 2B,C shows the continuous coverage of Ti3C2 MXene on the electrode surface, that the thickness of films was less than 10 nm, and that the root mean square surface roughness was about 1.29 nm, corresponding to a single or few Ti3C2 MXene layers [34]. SEM images in Figure S2 showed a Ti3C2 cluster with the typical multilayer structure after HF exfoliation. Figure 2D revealed the typical morphology of a few layers of Ti3C2 MXene with a wrinkled paper-like surface, which could provide potential sites for

Characterization of Ti 3 C 2 MXene
The X-ray diffraction (XRD) features of Ti 3 AlC 2 and Ti 3 C 2 MXene are shown in Figure 2A. The (002) peak of Ti 3 C 2 MXene shifted to a lower angle as compared to the commercial Ti 3 AlC 2 phase, suggesting a successful removal of Al layers after HF exfoliation [33]. The appearance of the Ti 3 C 2 MXene peak at approximately 25 • was attributed to the (104) diffraction of TiO 2 , which demonstrated a certain amount of oxides. The morphology and structure of Ti 3 C 2 MXene film were characterized by atomic force microscope (AFM) ( Figure S1A,B) and scanning electron microscopy (SEM) ( Figure 2D). Figure 2B,C shows the continuous coverage of Ti 3 C 2 MXene on the electrode surface, that the thickness of films was less than 10 nm, and that the root mean square surface roughness was about 1.29 nm, corresponding to a single or few Ti 3 C 2 MXene layers [34]. SEM images in Figure S2 showed a Ti 3 C 2 cluster with the typical multilayer structure after HF exfoliation. Figure 2D revealed the typical morphology of a few layers of Ti 3 C 2 MXene with a wrinkled paper-like surface, which could provide potential sites for effective ion and electron transport [35]. In order to explore the interaction between Ti 3 C 2 MXene and aptamers, zeta potential was applied to investigate the charged states of Ti 3 C 2 MXene and aptamer/Ti 3 C 2 MXene as shown in Figure 2C, which indicated that they were all negatively charged and their com-bination may not be caused by electrostatic reaction [36]. To further validate the binding method between the aptamer and Ti 3 C 2 MXene, X-ray photoelectron spectroscopy (XPS) was employed to analyze the elemental components and chemical states of both Ti 3 C 2 MXene and the aptamer/Ti 3 C 2 MXene. As presented in Figure 2D, the characteristic peaks of Ti 2p (455.8 eV), C 1s (284.8 eV), O 1s (530.5 eV), and F 1s (685.3 eV) elements appeared in the wide spectra of the Ti 3 C 2 MXene and aptamer/Ti 3 C 2 MXene. The appearance of P 2p (133.5 eV) and the increase in the percentage content of oxygen (19.13% to 41.36%) indicated the effective combination of Ti 3 C 2 MXene and aptamer. The peak splitting spectrum of Ti, C, O, F, and P shown in Figure S3 further proved the effective synthesis of Ti 3 C 2 MXene and the successful combination of Ti 3 C 2 MXene and aptamer. Specifically, the Ti 2p XPS spectrum of Ti 3 C 2 MXene was separated into Ti 2p 3/2 and Ti 2p 1/2 spin-orbit doublets, which showed five valence states ascribed to Ti (455. , which were assigned to 529.9, 530.8, and 531.9 eV, respectively [37]. After being modified with an aptamer, the peak of Ti 2p spectra corresponding to the Ti 2+ at 463.2 eV disappeared. Furthermore, the P 2p spectrum exhibited two peaks; the typical peak was attributed to the phosphate group (PO 4 − ), and the other may be related to the chelation interaction between the PO 4 − and Ti 2+ [38].
Molecules 2023, 28, x FOR PEER REVIEW 4 of 13 effective ion and electron transport [35]. In order to explore the interaction between Ti3C2 MXene and aptamers, zeta potential was applied to investigate the charged states of Ti3C2 MXene and aptamer/Ti3C2 MXene as shown in Figure 2C, which indicated that they were all negatively charged and their combination may not be caused by electrostatic reaction [36]. To further validate the binding method between the aptamer and Ti3C2 MXene, X-ray photoelectron spectroscopy (XPS) was employed to analyze the elemental components and chemical states of both Ti3C2 MXene and the aptamer/Ti3C2 MXene. As presented in Figure 2D, eV, respectively [37]. After being modified with an aptamer, the peak of Ti 2p spectra corresponding to the Ti 2+ at 463.2 eV disappeared. Furthermore, the P 2p spectrum exhibited two peaks; the typical peak was attributed to the phosphate group (PO4 − ), and the other may be related to the chelation interaction between the PO4 − and Ti 2+ [38].

Electrochemical Characterization of the Aptasensor Assembly Process
The change in the redox peak current (I P ) in cyclic voltammetry (CV) is related to the electron transfer rate constant and thus the electron transfer resistance at different modification steps. The electrochemical measurements were performed in 5 mM [Fe(CN) 6 ] 3−/4− at a scan rate of 0.1 V/s over a potential range of −0.4 V-0.8 V, respectively. CV for bare GCE, Ti 3 C 2 MXene/GCE, aptamer/Ti 3 C 2 MXene/GCE, Bovine serum albumin (BSA)/aptamer/Ti 3 C 2 MXene/GCE, and CAP/BSA/aptamer/Ti 3 C 2 MXene/GCE incubated electrodes are plotted in Figure 3. It can be seen from the black line and the red line that the current response of the electrode modified with Ti 3 C 2 MXene was higher than that of the bare GCE. This was because of the high conductivity of Ti 3 C 2 MXene, which could enhance the electron transfer rate. Subsequently, when Ti 3 C 2 MXene/GCE was modified with an aptamer, it resulted in a decreased I P (blue line) due to the negatively charged phosphate backbone in the aptamer that has a strong electrostatic repulsion on [Fe(CN) 6 ] 3−/4− [39]. The excess active site was occupied with BSA to avoid nonspecific adsorption, and a continued decrease in the current was observed (green line), indicating that the modified BSA inhibited electron transfer to the electrode surface. The I P decreased after the aptasensor was incubated with CAP (purple line), which could be ascribed to the formation of a complex, thereby obstructing electron transfer to the electrode surface.
GCE. This was because of the high conductivity of Ti3C2 MXene, which could enhance the electron transfer rate. Subsequently, when Ti3C2 MXene/GCE was modified with an aptamer, it resulted in a decreased IP (blue line) due to the negatively charged phosphate backbone in the aptamer that has a strong electrostatic repulsion on [Fe(CN)6] 3−/4− [39]. The excess active site was occupied with BSA to avoid nonspecific adsorption, and a continued decrease in the current was observed (green line), indicating that the modified BSA inhibited electron transfer to the electrode surface. The IP decreased after the aptasensor was incubated with CAP (purple line), which could be ascribed to the formation of a complex, thereby obstructing electron transfer to the electrode surface.
Nyquist plots of impedance spectra for different modification steps at GCE provide detailed information on the charge transfer mechanisms in the modification process, as shown in Figure 3B. The diameter of the semicircle in the Nyquist plot determines the charge transfer resistance of the electrode surface, and larger semicircles are obtained when there are slow electron transfer kinetics. The bare GCE exhibited a large semicircle domain (black line), implying a very large electron transfer resistance. After the electrode was assembled with Ti3C2 MXene, the resistance significantly decreased (red line) as a result of improved conductivity by facilitating more electron transfer to the electrode interface. The immobilization of the aptamer induced an increase in resistance (blue line) due to the negatively charged phosphate backbone in the aptamer. This acted as an electrostatic barrier and repelled [Fe(CN)6] 3−/4− anions, which retarded the electron transfer to the electrode surface. Then, the resistance increased upon incubation with BSA (green line) and increased upon incubation with CAP (purple line) due to the reduced flow of electrons from the solution to the surface of the electrode. These electrochemical impedance measurement (EIS) results were complementary to CV, thereby indicating the successful fabrication of the aptasensor.  Nyquist plots of impedance spectra for different modification steps at GCE provide detailed information on the charge transfer mechanisms in the modification process, as shown in Figure 3B. The diameter of the semicircle in the Nyquist plot determines the charge transfer resistance of the electrode surface, and larger semicircles are obtained when there are slow electron transfer kinetics. The bare GCE exhibited a large semicircle domain (black line), implying a very large electron transfer resistance. After the electrode was assembled with Ti 3 C 2 MXene, the resistance significantly decreased (red line) as a result of improved conductivity by facilitating more electron transfer to the electrode interface. The immobilization of the aptamer induced an increase in resistance (blue line) due to the negatively charged phosphate backbone in the aptamer. This acted as an electrostatic barrier and repelled [Fe(CN) 6 ] 3−/4− anions, which retarded the electron transfer to the electrode surface. Then, the resistance increased upon incubation with BSA (green line) and increased upon incubation with CAP (purple line) due to the reduced flow of electrons from the solution to the surface of the electrode. These electrochemical impedance measurement (EIS) results were complementary to CV, thereby indicating the successful fabrication of the aptasensor.

Optimization of Experimental Conditions
To improve the analytical performance of the constructed electrochemical aptasensor, the experimental conditions, including the concentration and volume of Ti 3 C 2 MXene, the concentration of aptamer, the incubation time of aptamer, and the incubation time of CAP, were optimized, as shown in Figure 4. As seen in Figure 4A, with the increase in the Ti 3 C 2 MXene concentration in the range of 0-0.25 mg/mL, the I P became higher, which was attributed to the fact that more electronic channels were provided to accelerate the transfer of electrons. However, when the concentration of Ti 3 C 2 MXene was higher than 0.25 mg/mL, the excess Ti 3 C 2 MXene would agglomerate on the electrode surface to form an electronic barrier layer, thus reducing the I P . Thus, 0.25 mg/mL Ti 3 C 2 MXene was selected to fabricate the Ti 3 C 2 MXene/GCE. The volume of Ti 3 C 2 MXene was also investigated, shown in Figure 4B,C, in which the I P increased with the increasing volume at the beginning (0 to 8 µL). The maximum current was obtained with 8 µL of Ti 3 C 2 MXene suspension modified electrode, and the I P decreased as the volume further increased. Similarly, this might be caused by the excess amount of Ti 3 C 2 MXene possibly hindering the electron transfer. Hence, the aptasensor was prepared by 8 µL Ti 3 C 2 MXene suspension modified on the GCE surface for further study.

Analytical Performance of the Electrochemical Aptasensors
For the purpose of evaluating the performance of the prepared CAP aptasensor, the electrochemical responses of the sensors modified with different concentrations of CAP were measured by differential pulse voltammetry (DPV) under optimal conditions. In Figure 5A, it is noted that the IP decreased as the concentration of CAP increased. A linear relationship was found between IP and the concentration in the range of 10 fM-1 µM. The calibration curve is y = −0.107log10CCAP + 1.607 (Pearson's r = 0.995), and the limit of detection is 1 fM. In addition, the prepared aptasensor in this work was compared with previous reports, and the results are shown in Table 1. Compared with previous reports, the aptasensor has the merits of being simple, low-cost, a wider linear range, and having a lower detection limit.   Figure 4D, the change in I P initially increased with the increasing concentration of the aptamer. When the aptamer concentration reached 0.5 µM, the change in I P reached its maximum value; subsequently, it gradually decreased. These observations suggest that the active sites on the surface of Ti 3 C 2 MXene/GCE reach a state of complete saturation when the aptamer concentration reaches 0.5 µM. As the aptamer concentration continues to increase, over-saturation of the aptamer at the electrode surface will hinder electron transfer between the electrode surface and [Fe(CN) 6 ] 3−/4− [39]. Therefore, a suitable concentration of 0.5 µM was selected for subsequent experiments. Immediately after, the influence of the aptamer incubation time on the aptasensor was studied, as shown in Figure 4E. The CAP aptamer was immobilized on the electrode surface and allowed to incubate for 1 h, 2 h, 3 h, 4 h, and 5 h, respectively. It was observed that the change in I P reached its maximum at 2 h and subsequently stabilized, which indicated that the aptamer reached its saturation point at 2 h. Therefore, 2 h was considered the optimal incubation time for aptamer immobilization in subsequent experiments. The incubation time of CAP is also an important factor affecting aptasensor performance, which was optimized, shown in Figure 4F. The change in I P reached its maximum value when the CAP incubation time was 40 min, and it was subsequently stabilized. These results suggest that a prolonged incubation time can provide a more sufficient reaction, bigger deviations of charge and resistance can be induce on an aptasensor, and then the increased I P can be measured. Thus, the optimal incubation time for CAP in subsequent experiments was selected as 40 min.

Analytical Performance of the Electrochemical Aptasensors
For the purpose of evaluating the performance of the prepared CAP aptasensor, the electrochemical responses of the sensors modified with different concentrations of CAP were measured by differential pulse voltammetry (DPV) under optimal conditions. In Figure 5A, it is noted that the I P decreased as the concentration of CAP increased. A linear relationship was found between I P and the concentration in the range of 10 fM-1 µM. The calibration curve is y = −0.107log 10 C CAP + 1.607 (Pearson's r = 0.995), and the limit of detection is 1 fM. In addition, the prepared aptasensor in this work was compared with previous reports, and the results are shown in Table 1. Compared with previous reports, the aptasensor has the merits of being simple, low-cost, a wider linear range, and having a lower detection limit. The reproducibility of aptamer/Ti3C2 MXene/GCE was evaluated by DPV analysis of five identically prepared electrodes ( Figure 5C). The relative standard deviation (RSD, n = 3) was 4.19% for CAP, showing that the aptamer/Ti3C2 MXene/GCE had very good reproducibility. The stability of the sensor was also tested by measuring the current responses for a week ( Figure 5D). For 100 nM CAP, the RSD was 5.26%, and the IP for CAP was reduced by 1.38% by the end of one week, depicting their acceptable stability. Generally, the aptasensor maintained its sensitivity for a week without significantly losing its current response. In order to evaluate the specificity of the developed electrochemical aptasensor, oxytetracycline (OTC), tetracycline (TET), doxycycline hydrochloride (DOX), streptomycin (STR), neomycin (NEO), and kanamycin (KANA) were used as interference substances. The effects of them on the current responses were assessed by DPV, as shown in Figure 5E. The response of the aptasensor to interference substances was significantly lower than CAP, indicating that the electrochemical responses were unaffected in the presence of potentially interfering antibiotics.

Detection of Chloramphenicol in Real Samples
In this experiment, the marked milk samples were taken as artificially contaminated milk and used for analysis of real samples so as to verify the feasibility of this method for detecting CAP in real food samples. The recovery rate of CAP was calculated based on the   The reproducibility of aptamer/Ti 3 C 2 MXene/GCE was evaluated by DPV analysis of five identically prepared electrodes ( Figure 5C). The relative standard deviation (RSD, n = 3) was 4.19% for CAP, showing that the aptamer/Ti 3 C 2 MXene/GCE had very good reproducibility. The stability of the sensor was also tested by measuring the current responses for a week ( Figure 5D). For 100 nM CAP, the RSD was 5.26%, and the I P for CAP was reduced by 1.38% by the end of one week, depicting their acceptable stability. Generally, the aptasensor maintained its sensitivity for a week without significantly losing its current response. In order to evaluate the specificity of the developed electrochemical aptasensor, oxytetracycline (OTC), tetracycline (TET), doxycycline hydrochloride (DOX), streptomycin (STR), neomycin (NEO), and kanamycin (KANA) were used as interference substances. The effects of them on the current responses were assessed by DPV, as shown in Figure 5E. The response of the aptasensor to interference substances was significantly lower than CAP, indicating that the electrochemical responses were unaffected in the presence of potentially interfering antibiotics.

Detection of Chloramphenicol in Real Samples
In this experiment, the marked milk samples were taken as artificially contaminated milk and used for analysis of real samples so as to verify the feasibility of this method for detecting CAP in real food samples. The recovery rate of CAP was calculated based on the added value and the detected value in the same solution. Trace amounts of CAP standard solutions (10 pM, 10 3 pM, 10 4 pM, and 10 5 pM) were added to the milk samples for detection. The detection data for the four samples are collected, respectively, in Table 2, with recovery rates ranging from 96.13% to 108.15% (n = 3), and the RSD varying from 2.637% to 8.342%. These results showed that the prepared electrochemical aptasensor based on Ti 3 C 2 MXene can reliably detect CAP in milk.

Apparatus
All electrochemical measurements were conducted using the conventional threeelectrode system, in which a GCE was employed as the working electrode (WE), a platinum wire was utilized as the counter electrode (CE), and a saturated Ag/AgCl electrode was used as the reference electrode (RE). Electrochemical experiments on CV and DPV were carried out with a CHI 660E electrochemical workstation (Beijing Huakoptian Technology Co., Ltd., Beijing, China). CV was recorded in the potential range of −0.2-0.8 V (vs. Ag/AgCl) and −0.2-0.6 V (vs. Ag/AgCl) in a 0.1 M PBS (PH 7.4) for the volume of Ti 3 C 2 MXene and concentration of Ti 3 C 2 MXene, respectively. Meanwhile, CV was recorded in the potential range of −0.4-0.8 V (vs. Ag/AgCl) in a 5 mM Fe(CN) 6 3−/4− electrolyte solution containing 0.1 M KCl for optimizing the other experimental conditions. DPV was measured in the potential range of −0.4-0.8 V (vs. Ag/AgCl) in a 5 mM Fe(CN) 6 3−/4− electrolyte solution containing 0.1 M KCl to determine the concentration of CAP.
The morphology of the prepared materials was characterized using SEM (S-3500 N, Hitachi, Tokyo, Japan) and AFM (Dimension Icon Bruker, Billerica, MA, USA), respectively. XPS was used for the element analysis, which was performed on an Axis Ultra DLD instrument (Kratos Analytical Ltd., Manchester, UK). XRD (Rigaku Co., Tokyo, Japan) was employed to analyze the structure of Ti 3 C 2 MXene. The Zeta potential values of Ti 3 C 2 MXene and aptamer/Ti 3 C 2 MXene were investigated utilizing a Zetasizer nano-particle potentiometer (Tianjin Xina Intelligent Technology Co., Ltd., Tianjin, China).

Synthesis of Ti 3 C 2 MXene
Ti 3 C 2 MXene was synthesized utilizing Ti 3 AlC 2 as the precursor by means of etching Al in HF solution ( Figure 1A) [47]. The synthesis steps were as follows: First, 10.0 g of Ti 3 AlC 2 powder was weighed and slowly added to 40% HF solution (100 mL) in a Teflon container. The mixture was stirred continuously at room temperature for 3 h and then diluted with deionized water (300 mL). The resulting diluted solution was centrifuged at 1467 RCF (relative centrifugal force) for 15 min. The sediment was collected and washed with deionized water until the PH reached 7.0. Then, the cleaned precipitate was vacuum dried at 100 • C for 6 h to obtain Ti 3 C 2 MXene multilayer film. Afterward, 2.5 g of dry powder was weighed and added into distilled water (400 mL). After ultrasonic treatment for 2 h, the supernatant was centrifuged (367 RCF) for 15 min, and the supernatant was filtered by 0.22 mm filter membrane. After drying at 100 • C for 24 h, Ti 3 C 2 MXene powder with single or fewer layers was obtained.

Construction of the Aptasensor
The procedure for making the aptasensor is illustrated in Figure 1B. Briefly, the GCE was polished with 0.3 µm and 50 nm alumina slurry until the surface of the electrode was specular sequentially, followed by thorough washing with ethanol solution and distilled water, respectively. Then, the prepared Ti 3 C 2 MXene suspension (0.25 mg/mL, 8 µL) was dropped on the polished GCE (Ti 3 C 2 MXene/GCE) and dried directly at room temperature.
After rinsing with the deionized water, the modified electrode was incubated with aptamer solution (0.5 µM) for 2 h in air (aptamer/Ti 3 C 2 MXene/GCE). Subsequently, the electrode was washed with deionized water to remove unbound aptamers. Due to the high affinity of Ti 3 C 2 MXene towards ssDNA, it can facilitate the attachment of aptamer onto the electrode surface. Subsequently, the aptamer/Ti 3 C 2 MXene/GCE was treated with 1% BSA (10 µL) to block non-specific binding sites and incubated at 4 • C for 1 h. After being thoroughly washed by deionized water several times, the prepared modified electrode (BSA/aptamer/Ti 3 C 2 MXene/GCE) was finally stored at 4 • C for subsequent experiments.

Milk Sample Measurement
Real samples were purchased from a local supermarket (Tianjin, China) and analyzed for CAP. Firstly, 10 mL of UP water was used to dilute 10 mL of milk sample. Then, 20% HAc was added to precipitate the protein, and the diluted milk sample was adjusted to pH 4.6. Centrifuging (9168 RCF) for 25 min to remove the precipitated protein and retain the supernatant. Then, adjust the pH to 7.0. Therewith, the sample was filtered by 0.22 µm filter membrane [48]. Finally, different concentrations of CAP (10 pM, 1 nM, 10 nM, and 100 nM) were added to the milk samples and detected by the proposed electrochemical aptasensor. According to the detection results, the recovery rate was calculated, and the practicability of the sensor was evaluated.

Conclusions
In conclusion, a simple, environmentally friendly, highly selective, and sensitive electrochemical aptasensor was developed for determining CAP in milk. The excellent performance of the sensor is attributed to the significantly increased electroactive surface area and conductivity resulting from the extraordinary properties of the Ti 3 C 2 MXene modifier. The proposed sensor manifests prime reproducibility, stability, and high selectivity with a detection range from 10 fM to 1 µM. The aptamer/Ti 3 C 2 MXene/GCE showed an acceptable recovery rate of 96.13-108.15% with an RSD of less than 9% for the electrochemical determination of CAP in milk. Therefore, the proposed electrochemical aptasensor has a high potential for real-time monitoring of CAP in food samples.